We propose to identify features in the human genome that define all potential origins (ORIs) of DNA replication and features that define which subset of all potential ORIs will be activated for initiation of DNA replication. The disagreement in prio results by others to map ORIs genome-wide may reflect contamination in nascent strand preparations that were enriched by lambda exonuclease (Lexo). Our preliminary results reveal that Lexo cannot digest G quadruplex (G4) structures; therefore, G4 will significantly contaminate nascent strand preparations. We will correct for this problem by normalization to a matched internal control and by use of buffer conditions that minimize Lexo biases. These corrections will be used in nascent strand sequencing (NS-seq) to map active ORIs genome-wide. Moreover, identification of the strand switch for leading strand synthesis will confirm the position of ORIs of bi-directional replication. We will first apply our revised NS-seq protocol to budding yeast where all ORIs are known in the genome, thus validating our revised NS-seq approach. Next, we will apply the revised NS-seq protocol to the genomes of three human cells lines, allowing us to compare active ORIs used by normal cells (GM06990) to those used in cancer cells (HeLa and MCF7). We will validate our results by use of the orthogonal approach of DNA combing for a few selected ORIs from our data set. DNA combing will also allow us to determine the spacing between active ORIs on single DNA molecules. Chromatin immunoprecipitation (ChIP) will be used to identify all potential ORIs in the genome (those that bind ORC2) and the subset that are active ORIs (those that bind Cdc45). For this, we will employ ChIP-reChIP approaches coupled with ChIP-exo. The Cdc45 binding sites at active ORIs will also serve to validate the active ORIs mapped by NS-seq. The significance of our experiments is two-fold. First, at the biological level, the sum of our results will allow correlatons to be drawn for features in the genome that correlate with the definition of all potential ORIs and the features that correlate with the subset of these that become active ORIs. Second, at the technical level, our corrections for Lexo biases will allow our development of revised protocols for NS-seq, thereby impacting the replication field.